Secreted IGFBP-3 can re-enter cells by endocytosis and translocate to the nucleus. It has been proposed that nuclear localization of IGFBP-3 is essential for it to be able to induce apoptosis. To test this hypothesis, we prepared plasmids in which IGFBP-3 lacking its signal prepeptide so that it would not be secreted was fused to yellow fluorescent protein (YFP). Fusion proteins were prepared with either wild-type IGFBP-3 or IGFBP-3 in which its nuclear localization signal (NLS) had been mutated. Following transfection of PC-3 human prostate cancer cells with plasmids expressing the YFP-IGFBP-3 fusion proteins, confocal microscopy showed that wild-type IGFBP-3 was predominantly localized to the nucleus whereas the IGFBP-3 NLS mutant was predominantly in the cytoplasm. Despite its cytoplasmic localization, the NLS mutant IGFBP-3 induced apoptotic cell death as determined by caspase-dependent Annexin-V staining quantified by flow cytometry. The same result was obtained when a mutation was introduced into the IGF-binding site of the IGFBP-3 NLS mutant. These results indicate that IGFBP-3 can induce apoptosis in human prostate cancer cells in an IGF-independent manner without being concentrated in the nucleus.